PROJECT SUMMARY/ABSTRACT PROJECT 1 Cardiac development relies on the stepwise activation and repression of lineage-specific gene expression programs. This process is regulated by conserved cardiac transcription factors (cTFs), such as NKX2.5, GATA4, TBX5, MEF2C and Myocardin, which cooperate with one another and chromatin-remodeling complexes to establish cellular identity by controlling gene regulatory networks. The critical nature of cooperative interactions is highlighted by a heterozygous glycine-to-serine missense mutation in GATA4 (GATA4-G296S) that disrupts interaction with TBX5 and causes congenital heart disease (CHD). TBX5 and GATA4 interact with the BAF complex of chromatin-remodeling proteins (investigated in Project 2), and together, these factors promote cardiac reprogramming in embryos. Further addition of MEF2C and its co- activator, Myocardin, (investigated in Project 3) reprograms adult cardiac fibroblasts into cardiomyocyte-like cells. Our Preliminary Data revealed that TBX5 and GATA4 interact with several nucleoporins that constitute the nuclear pore complex (NPC) at the nuclear membrane. Nucleoporins can control activation or silencing of developmental genes by regulating the three-dimensional chromatin architecture, but how specific genomic regions are recruited to the nuclear membrane remains unclear. Here, we will test the hypothesis that GATA4 and TBX5 interact in a lineage-specific fashion with NPC proteins to recruit genomic loci to the nuclear membrane to regulate the transcriptional output during cardiac differentiation. We will use hiPSC-derived CMs in which disruption of individual proteins or their interaction is possible using CRISPR/Cas9-based genome engineering (supported by Core C), and validate the findings in vivo in mice. We propose that the defective interaction between GATA4 and TBX5 disrupts the stoichiometry of the protein complex with nucleoporins (supported by Core A) and, thereby, contributes to the altered cardiac transcriptional and epigenetic outcome associated with disease (supported by Core B). Our specific aims are as follows: Aim 1) determine which nuclear pore proteins interact and co-localize with TBX5 or GATA4 to establish the lineage-specific three- dimensional genomic architecture in human cardiomyocytes and reprogrammed cardiomyocyte-like cells; Aim 2) determine the nature of enhancer elements localized to the NPC through interaction with TBX5 or GATA4, and the related epigenetic and transcriptional consequences during cardiomyocyte differentiation and reprogramming; and Aim 3) determine the effects of the human disease?causing mutation in GATA4 that disrupts interaction with TBX5 on the 3D genomic architecture, the transcriptional and epigenetic states of loci recruited to the nuclear membrane, and the interdependence of these events on physical interaction between GATA4 and TBX5. These studies represent a novel investigation into the role for lineage-specific TFs in regulating gene transcription by localization of specific genomic loci to the NPC and aim to open a new field in understanding the flow of genetic information during CM lineage specification.